Hippocampals neurogenesis is impaired in mice with a deletion in the coiled coil domain of Talpid3—implications for Joubert syndrome

Abstract Mutations in Talpid3, a basal body protein essential for the assembly of primary cilia, have been reported to be causative for Joubert Syndrome (JS). Herein, we report prominent developmental defects in the hippocampus of a conditional knockout mouse lacking the conserved exons 11 and 12 of Talpid3. At early postnatal stages, the Talpid3 mutants exhibit a reduction in proliferation in the dentate gyrus and a disrupted glial scaffold. The occurrence of mis-localized progenitors in the granule cell layer suggests a role for the disrupted glial scaffold in cell migration resulting in defective subpial neurogenic zone-to-hilar transition. Neurospheres derived from the hippocampus of Talpid3fl/flUbcCre mouse, in which Talpid3 was conditionally deleted, lacked primary cilia and were smaller in size. In addition, neurosphere cells showed a disrupted actin cytoskeleton and defective migration. Our findings suggest a link between the hippocampal defects and the learning/memory deficits seen in JS patients.


Introduction
Primary cilia are highly conserved sensory organelles, which protrude from the cell surface like an antenna. They are non-motile and have an axoneme made of microtubules (9 + 0) and a basal body that acts as a microtubule organizing centre (1). Motor proteins facilitate the movements of numerous proteins to and from the primary cilium. In addition, the primary cilium acts as a specialized compartment with a unique environment and the presence of transmembrane receptors allows it to transduce extracellular signals (2). Primary cilia are essential for transduction of signals of the sonic hedgehog (Shh) pathway in vertebrates as lack of cilia leads to abrogation of Shh signalling (3).
There are a number of rare human conditions, in which cilia are disrupted collectively termed 'ciliopathies'. These syndromes often involve a number of organs such as the brain, limbs, kidneys and eyes (4,5). One such ciliopathy is Joubert syndrome (JS) (6,7). Several causative genes have been identified for JS and currently numbering at 34 (8). One recently identified gene with mutations in JS patients is KIAA0586 or TALPID33 (9)(10)(11)(12).
Talpid3 was first identified in the talpid chick mutant (13,14). Subsequently, it was found that the talpid chick embryos with a mutation in Talpid3 lack primary cilia leading to aberrant Shh signalling (15). Talpid3 is a centrosomal protein essential for the docking of the basal body to the cell membrane (16). The Talpid3 protein has a conserved coiled coil domain and rescue assays with expression vectors encoding this domain alone showed that it is essential but not sufficient to rescue the formation of cilia and Shh signalling in the neural tube of the Talpid3 chick (16). Talpid3 is conserved in vertebrates and is essential for cilia formation not only in the chick (16) but also in the mice (17) and zebrafish (18). Mice with a constitutive deletion of the conserved exons 11 and 12 of Talpid3 (2700049A03Rik) are embryonic lethal. Conditional deletion of these exons in the limb leads to polydactyly, which was attributed to loss of primary cilia and aberrant Shh signalling (17).
Neural defects seen in JS often include hydrocephaly and hypoplasia or aplasia of the cerebellar vermis and the classic molar tooth sign. Another phenotype commonly described in JS is intellectual disability (19). Hippocampal abnormalities such as malrotation have also been reported in a subset of JS subjects suggesting a potential link with the learning and memory deficits seen in these patients (20,21). This aspect of JS is not well studied mainly due to the paucity of viable mouse models that fully recapitulate the JS brain phenotype.
The mouse hippocampus consists of two distinct structures-the cornu ammonius (CA1, Ca2 and CA3) and the dentate gyrus (DG. The dentate gyrus of the hippocampus is one of the few regions of the CNS in which neurogenesis continues into adulthood (22)(23)(24). A number of signalling pathways regulate the development of the dentate gyrus. One well studied pathway is the Shh pathway, which has been shown to be required for the proliferation of granule progenitors (25,26) and transduction of Shh signalling through the primary cilium has been shown to be important for hippocampal development. Conditional deletion of genes essential for Shh signalling, such as Smo, leads to abnormal development of neuronal precursors and causes hypotrophy of the dentate gyrus in mice (26). Mice lacking cilia components, such as Kif3a (a member of the kinesin family), Ift88 (intraf lagellar transport component) and B9d2 (basal body component) all show defects in adult hippocampal neural precursors (26,27).
We have generated mice with a conditional deletion of Talpid3 in the central nervous system by crossing the Talpid3 fl/fl mouse with a NestinCre deleter mouse strain (28). The Talpid3 fl/fl NesCre mice exhibited ataxia and examination of the brain showed a strikingly hypoplastic cerebellum with hall mark features of JS (29). Since primary cilia and Shh signalling play an important role in stem cell proliferation in the dentate gyrus and Talpid3 is essential for the formation of primary cilia, we examined the hippocampus of Talpid3 JS mice for defects that may have bearing on the hippocampal defects seen in JS patients.

Results
We undertook a detailed analysis of the hippocampus of the Talpid3 fl/fl NesCre (Talpid3 mutant) and the Talpid3 fl/fl (control) siblings between e18.5 and P15. The earliest stage at which the different regions of the mouse hippocampus can be clearly identified is e18.5. The pyramidal neurons of Ammon's horn complete their migration by P5 and the subpial neurogenic zone (SPZ) to hilar transition can be seen at this stage. These structures mature between P10 and P15 accompanied by growth and proliferation of the subgranular zone (SGZ) of the dentate gyrus. This is followed by a decrease in the rate of proliferation after P15. The oldest time point at which we investigated the hippocampus of the Talpid3 mutant mice was P15 due to the development of extreme ataxia at which point the mice have to be culled.

The Talpid3 mutant hippocampus lacks primary cilia
We immunostained sections of the brain from the Talpid3 mutant and control mice with antibody to Adenyl cyclase 3 (ASCIII) (30) to detect primary cilia ( Fig. 1). At P15, primary cilia were present in most cells in the predominantly quiescent granule cell layer (GCL) of the control dentate gyrus but were not detectable in the GCL of Talpid3 mutant mice ( Fig. 1A and B). Compared to control siblings, the SGZ of the Talpid3 mutant mice also showed a significant reduction (80%) in the number of cells with primary cilia. We also immuno-stained for primary cilia at e18.5, when the hilus, GCL and SPZ are identifiable in the dentate gyrus (Fig. 1C). At e18.5, primary cilia were clearly seen in the SPZ (Fig. 1D) of the sibling control mice but were absent in the Talpid3 (Fig. 1D).

The Talpid3 mutant hippocampus is misshapen with defects in the dentate gyrus
The morphology of the hippocampus at e18.5 was comparable between control and Talpid3 mutant mice but by early postnatal stages, defects in the hippocampus were noticeable. On first inspection, the P15 Talpid3 mutant hippocampus in H&E stained sections appeared more elliptical and compressed as compared to that in the control sibling mice, presumably a consequence of the hydrocephaly (Fig. 2A). The gross morphology of the dentate gyrus and Ammon's horn appeared similar to control sibling mice. However, upon closer examination, the dentate gyrus appeared thinner with a lower cell density (Fig. 2C). At P15, both the GCL and the SGZ of the dentate gyrus were thinner in the Talpid3 mutant ( Fig. 2D and E). The SGZ of the Talpid3 mutant mice also had a reduced number of darkly pigmented cells ( Fig. 2B and C), which have previously been identified as the progenitor population (26,31). The region below the SGZ also consistently exhibited a loss of tissue integrity which often resulted in tearing of the section ( Fig. 2B and C).

Organization of the Hippocampus
Mature axonal tracts were identified by immunolabelling for neurofilament (165 kDa). In horizontal sections of the P15 Talpid3 mutant brain, the hippocampus was seen to be more elliptical as compared to the control (Fig. 3A) and the dentate gyrus was smaller. Although the GCL displayed the characteristic 'U' shape, its thickness was reduced compared to the control GCL. Despite the reduced thickness of the dentate gyrus, both the CA1 and CA3 fields in the Talpid3 mutant were comparable in size to the control (Fig. 3A). However, structures caudal to the hippocampus such as the presubiculum (PrS) and parasubiculum (PaS) were smaller in the Talpid3 mutant (Fig. 3A).
Many of the key axonal connections in the hippocampus are established by P5. To assess the effect of the loss of Talpid3 on this, we analyzed sagittal sections of the P5 brain immuno-stained for neurofilament (165 kDa) (Fig. 3B). Axons from the perforant path terminate in the molecular layer (ML) above the GCL. Both control and Talpid3 mutant showed a similar density and branching of axonal tracts (Fig. 3C). Radial axons of the Talpid3 mutant CA1, likely to be pyramidal neurons, appeared to be similar to control CA1. (Fig. 3D). This suggests that pyramidal neurons and connections, both in/out of the hippocampus, appeared to develop normally. However, the dentate gyrus appeared smaller and underdeveloped at this stage in the Talpid3 mutant mice.

Progenitor and mature neurons are mislocalized in the Talpid3 mutant hippocampus
Neurogenesis is regulated by the sequential expression of transcription factors. In the developing and adult DG, proliferating cells are classified as type1, type 2 and type 3 based on the transcription factors they express. Type 1 cells are radial glial cells (neural stem cells-NSCs) and express Pax6, GFAP and Sox2. Type 2 cells express Tbr2 and are the transit amplifying cells or the intermediate progenitors and the Type3 cells are the committed neuroblasts that express NeuroD (31,32).
Progenitor and mature neurons of the hippocampus were identified by staining for Pax6 and NeuN respectively during the development of the dentate gyrus (E18.5, P5, P10 and P15) in the Talpid3 mutant and control mice (Fig. 4). We found that the outer boundary of the GCL in the Talpid3 mutant was less well defined when compared to the control mice at P10. At this stage, we also observed a 2.4 fold increase in the number of NeuNpositive cells mis-localized in the ML (Fig. 4A, B and D). Talpid3 mutants also had a significantly fewer numbers of Pax6 positive progenitors in the developing SGZ ( Fig. 4A-C), 37% and 34% at P10 and P15, respectively (Fig. 4C). The other unexpected finding was the incidence of ectopic Pax6-positive progenitors in the GCL of the Talpid3 mutant ( Fig. 4B and C). At younger stages between E18.5-P5 significant numbers of progenitors were present in the control GCL, reflecting the cell migration in the SPZ-to-hilar transition (30). However, this was not the case at later stages. Quantification of progenitor numbers from three control and three Talpid3 mutant mice at P15, revealed that the Talpid3 mutants had a 6.1 fold higher number of Pax6-positive progenitors in the GCL compared to their sibling controls (Fig. 4C).

The Talpid3 mutant hippocampus exhibits a loss of radial glial progenitors and intermediate progenitors
NSCs can exist in an activated state or inactivated state and the minichromosome maintenance protein 2 (MCM2) can be used to distinguish these two states. Ki67, PCNA (proliferating cell nuclear antigen), and MCM2 are endogenous markers that we used to label the status of NSCs. MCM2 is expressed during all phases of the cell cycle but is downregulated when cells exit the cell cycle into a quiescent state as for example it has been shown that quiescent intestinal stem cells downregulate MCM2 (33). The nuclear protein Ki67 is expressed through the cell cycle but not in the G0 or early G1 phase (34). Similar to Ki67, cells in late G1, S and G2-M phases can be identified by the expression of Proliferating cell nuclear antigen (PCNA). Both these markers are associated with actively proliferating cells. In contrast, expression of MCM2 identifies all activated cells, i.e. cells leaving G0 to go to G1 phase, which means it identifies cells with proliferative potential (35,36,37). The Mcm2/Ki67 ratio, can therefore be used to estimate the population of cells that are in early G 1 (licensed to proliferate).
To determine the number of cycling cells in the hippocampus, Bromodeoxyuridine (BrdU) was administered to P15 mice 2 h before euthanasia and stained for BrdU+ve nuclei. Talpid3 mutants showed a 70% reduction in the number of cycling cells in the SGZ ( Fig. 5A and B). At this stage, only a low number of BrdU-positive cells were identified in both control and mutant GCL and a significant difference was not observed (Fig. 5B).
Intermediate progenitors (type 2b) and proliferating cells were identified by labelling for Tbr2 and Proliferating cell nuclear antigen (PCNA), respectively in the P10 dentate gyrus (Fig. 5C). Proliferating cells which are PCNA-positive RGC (type-1, Tbr2 negative) or non-radial glial progenitors (type-2a, Tbr2 positive). The Talpid3 mutant dentate gyrus showed a reduction   S1D and E) also did not show any statistical difference in the progenitor numbers. The similarity between control and Talpid3 mutant dentate gyrus progenitors was confirmed by staining the E18.5 dentate gyrus for additional proliferation markers PCNA and PH3 (Supplementary Material, Fig. S1D). Consistent with previous observations, the density of cycling cells and their level of proliferation was very comparable. Closer examination of sections stained with Pax6 and NeuN also exhibited little difference between control and Talpid3 mutant as progenitors were clearly present in the SPZ and GCL (Supplementary Material, Fig. S1E) at this stage. This suggests that, despite loss of primary cilia in the Talpid3 mutant at E18.5, the defective proliferation in the hippocampus is evident only in the postnatal stages.
We further quantified cells in the post-natal hippocampus of Talpid3 mutant and control siblings which were immuno-stained for MCM2 and Ki67 at P5, P10 and P15 ( Fig. 6A-E). Talpid3 mutants exhibited a significant decrease (51%) in the total number of proliferating (Ki67 positive) cells in the P5 SGZ but no reduction was seen in the number of MCM2 positive progenitors when compared to control (Fig. 6A, D and E). The total number of proliferating cells was also significantly reduced (45%) in the SGZ of the Talpid3 mutant at P10. In addition, although not significant, there appeared to be a reduction in the number of MCM2-positive progenitors (Fig. 6B, D and E). This difference in the SGZ became more apparent at P15, with Talpid3 mutants exhibiting a 63% reduction in MCM2-positive progenitors and 71% reduction in Ki67-positive proliferating cells compared to the control siblings (Fig. 6C, D and E).
To better assess the progenitor population in the Talpid3 hippocampus, the number of cells which were single or double-labelled for MCM2 and Ki67 were quantified at P5, 10 and 15. Compared to control siblings, Talpid3 mutants were found to have a greater proportion of non-dividing but activated SGZ progenitors (MCM2positive, Ki67-negative) (Supplementary Material, Fig.  S2A and B). This meant that in addition to having fewer MCM2-positive progenitors in the SGZ, fewer of these were also less able to actively proliferate leading to a depleted progenitor pool.
GFAP is used as a marker of radial and non-radial glial progenitors (type 1 and 2a), but it is also present in astrocytes of the dentate gyrus. By double labelling for GFAP and Ki67 it is possible to preferentially identify the proliferating glial progenitor population (Supplementary Material, Fig. S2C and D). Quantification of the proportion of Ki67-positive progenitors that were GFAP positive or GFAP negative demonstrated that there was no statistical difference between the proportions of glial and non-glial progenitors in the P15 SGZ when comparing control and Talpid3 mutant (Supplementary Material, Fig. S2E). Given that the total number of Ki67-positive cells was reduced, we concluded that there is a decline in the number of both glial progenitors and intermediate progenitors in the SGZ. This is the likely to be the cause of the hypoplasia seen in the GCL at later stages in the Talpid3 mutant mice.
Despite the progressive depletion of progenitors seen in the mutant SGZ, the total number of Ki67-positive cells appeared to show a slight increase from P10 onwards (Supplementary Material, Fig. S2D and E). Although these data were not significantly different, they follow the same trend seen with the numbers of Pax6-positive cells in the Talpid3 mutant GCL at this age (Fig. 4C). By P15 the total numbers of MCM2-positive and

Mislocalized progenitors correlate with poorly formed cellular scaffolds
Doublecortin (Dcx) is a cytoskeleton component closely associated with neurogenesis and migration (38,39). In order to explain the presence of misplaced progenitors and mature neurons in the Talpid3 mutant dentate gyrus, the distribution of Dcx in the dentate gyrus was analyzed between E18.5 and P15 ( Fig. 7A-C). The expression of Dcx in the control dentate gyrus at e18.5 was very low and was hard to detect immunohistochemically. The levels of Dcx increased at P5, with the strongest expression at the boundary of the GCL and ML (Fig. 7A). At this stage, Dcx expression was similar between the control and Talpid3 mutant sibling mice. However, in the P10 dentate gyrus of control sibling mice, there was an increase in the level of Dcx in the SGZ (Fig. 7B). Dcx-positive fibres seen spanning the GCL towards the ML in the control mice was not observed in the Talpid3 mutant SGZ or GCL.
The levels of Dcx expression in SGZ of the control mice increased further and at P15 was evident as a thick band labelling the SGZ (Fig. 7C). The Dcx positive fibres stretching through the GCL were also more obvious and many could be traced from SGZ through to the ML. In contrast, the Talpid3 mutant showed a striking absence of the thick Dcx band in the P15 SGZ and this correlated with the loss of tissue integrity (indicated by solid line, Fig. 7C). Dcx fibres stretching through the Talpid3 mutant GCL at P15 were also far less distinct. Although the distribution of Dcx in the Talpid3 mutant demonstrated a prominent phenotype, it seemed to correlate more with the loss of progenitors in the SGZ. Given the ectopic progenitors seen in the Talpid3 mutant GCL, it would seem reasonable to expect increased Dcx positive fibres, however, this was not observed at the later stages.
Many of the cell movements in the developing hippocampus are thought to be directed by glial-guided migration along radial fibres. The glial scaffold was assessed at P5 by co-labelling for GFAP and nestin where double-positive cells represent radial glial-progenitors (type 1 and type 2a) (Fig. 8A) (40). In the control dentate gyrus, both GFAP-positive and nestin positive fibres extended radially through the GCL and branch extensively in the ML. We observed two types of fibres; thicker primary fibres with simple radial organization, which then bifurcated into thin diffuse fibres with widespread branching. Upon closer examination, all visible fibres shared some co-localization of both GFAP and nestin (Fig. 8B). In the thicker primary fibres, GFAP had a more continuous distribution, whereas nestin was seen often intermittently along the length of the fibre. This high level of co-localization is a result of the large number of radial glial progenitors present at this age. The Talpid3 mutant dentate gyrus showed a dramatic loss of both types of fibre (Fig. 8B ). Most obvious was the loss of fine branches in the ML. The reduction in the numbers of primary fibres is likely to represent a loss of RGC progenitors. In addition, the loss of fine branching suggests a defect in the glial cell morphology.
It is likely that the glial defect contributes to the ectopic progenitors seen in the Talpid3 mutant GCL. To test this, sections of brain were co-immunostained for GFAP and the progenitor marker Pax6. At E18.5,  The Talpid3 mutant dentate gyrus at P5 exhibited a striking reduction in the occurrence of glial fibres and extent of their branching but showed no difference in the number of progenitors present in the GCL (Supplementary Material, Fig. S3B). Type 1 and type-2b progenitors were identified by immunostaining for Sox2 in addition to GFAP (Fig. 9A-C). In the Talpid3 mutant, many of the progenitors migrating through the GCL were not obviously associated with glial fibres and in addition several glial fibres appeared overloaded with progenitors on a single fibre (Fig. 9C). This is in contrast to the control mice, which had similar numbers of progenitors in the dentate gyrus but were better distributed through the glial scaffold. It is possible that the disrupted scaffold in the Talpid3 mutant dentate gyrus limits access to glial fibres or restricts their pathway to the SGZ. This in-turn could delay or inhibit their SPZ-to-hilar transition resulting in ectopic progenitors evident in the GCL from P10.
Reelin, an extracellular protein expressed by Cajal-Retzius cells in the ML plays an important role in the migration of hippocampal neurons and their dendritic branching (41). Reelin has been shown to be important in the movement of progenitors away from the SPZ towards the SGZ (41). It has also been demonstrated that Reelin is required for the correct formation of the RGC scaffold (38,42) and migration of post-mitotic granule neurons born in the SGZ (43)(44)(45). In the control P5 dentate gyrus, Reelin producing cells were clearly seen at the outer edge of the ML (Supplementary Material, Fig. S4). In the Talpid3 mutant dentate gyrus Reelin producing cells were also present at the edge of the ML. However, there appeared to be a slight reduction in the number of Reelin-positive cells, which was most evident in the posterior region above the dorsal blade of the dentate gyrus (Supplementary Material, Fig. S4). This defect may be a contributing factor to the disrupted migration seen by granule progenitors or post-mitotic granule neurons.

Talpid3 and cell migration
Many of the phenotypes that we observed in the Talpid3 mutant hippocampus are due to the complex interactions between the migrating cells and the glial scaffold. We took an in vitro approach to begin to address the questions arising from our findings on the Talpid3 mutant mouse hippocampal phenotype by using hippocampal neurosphere cultures ( Supplementary  Material, Fig. S5). Hippocampal neurosphere lines were generated from postnatal P5 mice obtained by breeding Talpid3fl/fl;UbcCreER T2 males to Talpid3 fl/fl females as described in Materials and Methods. The expression of Cre in the UBcCreER T2 mouse driver line can be induced by administering 4-hydroxytamoxifen (4-OHT). Each neurosphere line was created from the hippocampus of an individual mouse. In total, 13 independent hippocampal neurosphere lines each of Talpid3fl/fl;UbcCreER T2 and Talpid3 fl/fl genotype were generated. Neurosphere lines were expanded, disassociated (46) and frozen. This allowed all experiments to be performed using cells of identical passage number from frozen stocks. In total, three independent neurosphere lines of genotype Talpid3fl/fl;UbcCreER T2 were selected for experimental analysis (designated '#1, 2, 3') (Supplementary Material, Fig. S5). In addition, neurosphere lines from Talpid3 fl/fl mice were also used as controls in the experiments.
We found that treatment of neurospheres with two doses of 4-OHT, one immediately after disassociation (0 day) and one 3 days later (3 days) led to robust recombination of Talpid3. The DNA from Talpid3fl/fl;UbcCreER T2 neurospheres treated with and without 4-OHT for 7 days growth were analyzed by PCR for the recombination event ( Supplementary Material, Fig. S6) showed a consistently high level of recombination caused by 4-OHT administration. Using this approach, we studied the consequences of Talpid3 loss from postnatal hippocampal progenitors using the experimental plan illustrated in Supplementary Material, Figure S5.

Neurospheres with a targeted deletion of Talpid3 exons 11 and 12 formed smaller colonies
The Talpid3 mutant hippocampus exhibited a loss of proliferating progenitors. To assess the consequences of the deletion in Talpid3 in the neurosphere lines we first monitored the effect on the growth of the neurospheres. Talpid3 fl/fl UbcCreER T2 neurospheres with and without with 4-OHT treatment were cultured for 7 days. Loss of Talpid3 resulted in smaller neurospheres (Fig. 10A).
One problem encountered with neurosphere culture is that free-floating cultures often fuse resulting in size heterogeneity. To limit this effect and study neurosphere growth, disassociated neurosphere cells were plated in 96-well tissue culture plates at low density. Cells proliferated and grew as a round adherent colony derived from a single cell (Fig. 10B). Measurement of the area of the colony from multiple wells showed that loss of functional Talpid3 resulted in significant reduction in median colony area in 4-OHT treated Talpid3 fl/fl UbcCreER T2 derived colonies as compared to untreated Talpid3 fl/fl UbcCre cells (Fig. 10C). An important control included the use of Talpid3 fl/fl cells with and without 4-OHT administration, which showed no difference in colony size. This confirms that the defect seen was due to loss of functional Talpid3 rather than any adverse effect of 4-OHT. To assess the long term effect of the deletion in Talpid3, neurospheres cultured with and without 4-OHT administration were disassociated after 7 days and plated on 96-well tissue cultures plates (named 'passage +1') (Fig. 10C). These cells did not have any further addition of 4-OHT, yet colonies consistently showed a significant reduction in median colony size when compared with treated and untreated controls. This suggests an intrinsic and permanent defect caused by the deletion in Talpid3.

Inducible deletion of exons 11-12 of Talpid3 in differentiated cells leads to loss of cilia and a disrupted actin cytoskeleton
We assessed the consequence of deletion of exons 11 and 12 in Talpid3 on primary cilia in differentiated cells derived from neurospheres by immunostaining for ACIII and acetylated α-tubulin. Cells with a deletion in Talpid3 had considerably fewer primary cilia ( Supplementary  Material, Fig. S7B). Primary cilia were detected on 61% of cells in neurospheres that had a functional Talpid3, whereas only 9% Talpid3 mutant neurosphere cells possessed a primary cilia (Supplementary Material, Fig.  S7C). This confirmed that deletion of exons 11 and 12 of Talpid3 had occurred in a significant number of cells which was sufficient to cause loss of primary cilia.
Previous reports have suggested that loss of Talpid3 leads to defects in the actin cytoskeleton (16,17). Staining with f luorescently labelled phalloidin was used to detect stabilized F-actin in differentiated cells generated from neurospheres as described in Materials and Methods (Supplementary Material, Fig. S8A), which showed a reduction in the number of stress fibres in cells with a deletion in Talpid3 (Supplementary Material, Fig. S8B). Importantly, however, increased levels of actin in the ruff led membrane or filopodia were not observed in these cells.

Deletion of exons 11 and 12 of Talpid3 affects migration of cells from neurospheres
One commonly used approach to monitor the capacity of neural cells to migrate is the neurosphere migration assay (47,48,49,50). In this paradigm, neurospheres of equal size are transferred to individual wells coated with Matrigel ® containing differentiation media. Migration of cells away from the neurosphere was monitored over the course of 72 h to provide a quantitative readout of neural migration (Fig 11A). Due to the nature of this assay, it is a useful system to monitor the fundamental ability of cells to migrate, rather than in response to signalling gradients. Outgrowths from both 4-OHT treated and untreated 7day Talpid3 fl/fl UbcCre neurospheres from three independent neurosphere lines were measured after 2, 24, 48 and 72 h of culture. At all the time points, OHT-treated cells had migrated significantly less distance than the untreated controls (Fig. 11B). In addition to the smaller radial area, the leading edge of the treated cultures had a lower density of cells after 48 h and this was maintained after 72 h ( Fig. 11C and D).

Discussion
We have previously shown that conditional deletion of the exons 11 and 12 of Talpid3 in the CNS leads to very severe cerebellar defects reminiscent of JS (29). Here we show that in addition to the cerebellar defects, the Talpid3 mutant mice exhibit prominent defects in the dentate gyrus. The Talpid 3 mutant hippocampus shows a reduction in the numbers of progenitors in the SGZ and ectopic progenitors in the GCL, which correlates with aberrant formation of the underlying glial scaffold. Here we show that defects occur in the SPZ-to-hilar transition in the hippocampus in addition to the proliferative defects described in other cilia deficient mutant mice.
The defective proliferation and aberrant progenitor localization in the Talpid3 mutant dentate gyrus is somewhat similar to that seen in the hippocampus of B9d2 fl/fl NesCre mice (27). Breunig et al. (27) found that the dentate gyrus of the B9d2 mutant mice at P0 was smaller with reduced proliferation. The Talpid3 mutant dentate gyrus appears to be normal at e18.5, and the first signs of defects were evident at P0 similar to the B9d2 mutant. It appears that the earliest stage of dentate gyrus development requiring Talpid3 is P0. In contrast, loss of Kif3a using the hGFAP-Cre deleter (Kif3a fl/fl hGFAPCre) resulted in a smaller dentate gyrus with reduced proliferation at e18.5 (26).
Both NesCre and hGFAP-Cre deleters have been shown to cause widespread recombination from mid-gestation onwards (28,51,52). It is likely that the defect in Talpid3 fl/fl NesCre dentate gyrus was not observed at an earlier stage because of the slightly different times of onset of recombination mediated by the hGFAP-Cre and NesCre deleter strains. However, it is also tempting to speculate that this difference in phenotype may be due to the differing roles of Talpid3 and Kif3a. It is plausible that the earlier phenotype seen in Kif3a fl/fl hGFAPCre dentate gyrus compared to the Talpid3 fl/fl NesCre and B9d2 fl/fl NesCre dentate gyrus may be due to a cilia-independent role of Kifa3a in the processing of Gli3.
The onset of defects in the dentate gyrus in the Kif3a fl/fl hGFAPCre occurs earlier than in the Talpid3 fl/fl NesCre and B9d2 fl/fl NesCre mice but all three mutants exhibit defective proliferation in the dentate gyrus (26,27). Han et al. extended this further and showed that there was a reduction in proliferation in the dentate gyrus of a hypomorphic IFT88 mutant (IFT88orpk/orpk) and Ftm (Rpgrip1l) −/− mice.
In the Talpid3 mutant dentate gyrus, there is loss of RGC (type 1), non-radial glial progenitors (type 2a) and intermediate progenitors (type 2b) resulting in a depletion of the progenitor pool similar to that seen in the B9d2 fl/fl NesCre mice that showed a loss of slow cycling progenitors and greater exit from the cell cycle (27). In the case of both B9d2 and Kif3a mutant mice, the defect in proliferation was attributed to loss of Shh signalling. The hippocampus in theTalpid3 mutant mice lack cilia resulting in a loss of Shh signalling. This is consistent with a loss of proliferation seen in Smo fl/-NesCre dentate gyrus (25). However, the phenotype in Smo fl/-NesCre dentate gyrus was more prominent with increased hypoplasia in the GCL. An even more severe phenotype was seen in the Gli3 fl/fl Emx1Cre mice, in which the initial blade of the dentate gyrus failed to form by E18.5 (52).
The Talpid3 mutant dentate gyrus had ectopic progenitors in the GCL, which is also seen in the dentate gyri of B9d2 fl/fl NesCre and Kif3a fl/fl hGFAPCre (26,27). Here, for the first time, we draw a causal link between the two phenotypes and suggest that progenitors are mislocalized as a direct result of the defective glial scaffold. In addition, in the Talpid3 mutant hippocampus, fewer progenitors were associated with radial fibres and those that were associated appeared to be overloaded. These observations suggest that the glial scaffold is insufficiently developed in the Talpid3 mutant hippocampus and is inadequate for SPZ-to-hilar migration. It is also important to note that between the ages of P10 and P15 the Talpid3 mutant showed a reduction in the number of ectopic progenitors in the GCL indicating that there may be a delayed or ineffective migration rather than a complete block.
We suggest a link between the glial scaffold abnormalities and defective progenitor migration in the Talpid3 mutant mice; however, the exact contribution of radial glia in progenitor migration is still unclear. To address this, studies specifically targeting the glial scaffold in precise time windows during development are needed to explicitly show the role and requirement of radial glia for the SPZ-to-hilar transition. It is also unclear whether loss of Talpid3 affects the glial branching directly or whether there is simply a reduction in the number of glial progenitors. Further studies looking at glial behaviour in vitro will help determine whether Talpid3 is directly required for glial scaffold formation. The severe hydrocephaly and ataxia seen in Talpid3 mutant mice precluded the study of the dentate gyrus at later stages using the NesCre deleter strain.
Reelin is a secreted protein, which is well known to influence the migration of neurons in the hippocampus. Talpid3 mutant dentate gyrus showed a subtle reduction in the number of Reelin-positive cells in the ML. Mice with constitutive loss of Reelin, the so-called 'Reeler' mice, have a disruption of hippocampus with aberrant radial glial scaffold (38,43) and failure of the SPZ-tohilar transition (42). Loss of Reelin signalling has also been shown to cause ectopic localization of post-mitotic granule neurons both in the hilus and ML (44,45). Reeler mutant mice also have a thinner entorhinal performant pathway termination zone in the ML (44). Studies of other primary cilia mutants have described relatively normal distribution of Reelin-positive cells in the marginal zone of the cortex of Kif3a fl/fl;hGFAPCre (53), Arl13b hnn/hnn (54) and hypomorphic Ift88 cbs/cbs (55) mice. The wide ranging defects seen in mutants with complete loss of Reelin makes it difficult to interpret the effects of the subtle reduction in Reelin-positive cells in the Talpid3 mutant hippocampus.
The Talpid3 mutant dentate gyrus is characterized by the presence of ectopic post-mitotic granule neurons in the ML. Disc1, a protein known to interact with the basal body, has been shown to be required for primary cilia formation in cultured cells (56). Loss of DISC1 resulted in aberrant placement of granule neurons in the adult hippocampus similar to that seen in Talpid3 mutant mice (57,58).
Neurospheres generated from the Talpid3 fl/fl UbcCreER T2 mice in which Talpid3 could be deleted in an inducible manner by addition of 4-OHT allowed us to dissect the role of Talpid3, if any, on growth of neurospheres and migration of cells. Neurospheres in which Talpid3 was deleted, lacked primary cilia and formed smaller colonies. In addition, they had a disrupted actin cytoskeleton and the cells showed a marked decrease their ability to migrate. The reduction in progenitor proliferation is consistent with the loss of dividing progenitors seen in the Talpid3 mutant hippocampus. It is likely that the smaller colony size seen in vitro and fewer hippocampal progenitors seen in vivo are regulated by the same mechanism.
To date, there have been a few studies looking at the effects of loss of primary cilia in hippocampal cultures. Breunig and colleagues (27) showed that, in slice cultures, loss of primary cilia rendered hippocampal progenitors unable to respond to exogenous Shh. In neurospheres derived from the perinatal SVZ, cooperation between Shh and EGF signalling was shown to regulate their formation and growth (59). Using culture conditions similar to the current study, the Hh inhibitor, cyclopamine, was able to reduce neurosphere proliferation indicating the occurrence of autocrine or paracrine Hh signalling within neurospheres. Given the known roles Shh in the hippocampus in vivo (25)(26)(27), this signalling pathway is a prime candidate inf luencing the growth of hippocampal neurospheres following loss of Talpid3.
Microtubules did not appear to be affected in the Talpid3 mutant neurospheres but had fewer F-actin stress fibres compared to controls. This is consistent with cytoskeletal phenotypes seen in other Talpid3 deficient cell types including talpid3 chick neural tube, cultured talpid3 chick limb cells (16) and Talpid3 flfl;PrrxCre mouse limb fibroblasts (17). Cells lacking IFT88 and basal body proteins BBS4, BBS6 and Mks3 have also been shown to have a disrupted actin cytoskeleton (61-63-56). The extent to which the disruption of actin cytoskeleton contributes to the hippocampal phenotype is unclear at present.
Cells from hippocampal neurospheres with a deletion in Talpid3 exhibited retarded migration. This suggests that there are defects in cell migration, which are independent of the glial scaffold since fewer cells were seen at the leading edge. Migration differences in this assay are likely to be the result of intrinsic defects in the ability of cells to move, rather than their ability to orientate themselves towards a morphogen. Our experimental data suggest that both loss of some intrinsic property of migrating cells and the defective underlying glial scaffold contribute towards the in vivo migratory defect and failure of SPZ-to-hilar transition.
Fibroblasts lacking Talpid3 have been described as having less directionality (17) in the scratch assay but we did observe any lack of directionality in cells migrating from the Talpid3 mutant neurospheres. Scratch assays have been used to study the role of cilial components in different cell types including endothelial cells and mouse embryo fibroblasts (MEFs) with hypomorphic IFT88 (60,63) and kidney cells lacking BBS4 or BBS6 (61,62). In all these cases, the cells lacked cilia and exhibited a reduced ability to migrate. MEFs have been shown to require primary cilia to transduce PDGFα signalling required for directional cell movement (64). It has been further shown that loss of IFT88 influences PDGFα signalling which has a resultant effect on MEK1/2-Erk1/2 pathway, ultimately affecting actin distribution at the lamellipodia of migrating fibroblasts (63). To what extent PDGFα influences cell migration in the neurosphere assay is not known but it has been shown that cells isolated from the hippocampus express components of the PDGFα signalling pathway (65).
In conclusion we have shown that the Talpid3 mutant mice have defects in the hippocampus, which exhibits reduced proliferation and ectopic progenitor cells. This may have a bearing in the learning and cognitive defects in JS patients. The Talpid3 mutant mice is an useful model to further unravel the molecular and cellular mechanisms underlying JS.

Colony management
Mice were maintained on a 12 h light/dark cycle with access to food and water ad libitum. All procedures were approved by the University of Bath Ethical Review Board and were conducted under HO animal procedures project (VS) and personal licences (AB and VS) in accordance with UK Home Office guidelines and the UK Animals (Scientific Procedures) Act, 1986.

Breeding to generate experimental mice
Talpid3 fl/fl mice were bred to NesCre mice to produce Talpid3 fl/wt;NesCre stud males. To obtain experimental embryos/mice, Talpid3 fl/wt;NesCre stud males were crossed to Talpid3 fl/fl female mice to generate Talpid3 fl/fl;NesCre mice that are referred to as Talpid3 mutant and Talpid3 fl/fl (control) mice.

Genotyping
Mice were genotyped using DNA isolated from tail biopsies or ear punches Genotyping was carried out by PCR using GoTaq ® Flexi DNA Polymerase (Promega, UK), according to manufacturer's instructions. Primer sequences used for genotyping of the different mouse strains are shown in Supplementary Material, Table S1.

Dissection and processing of tissues
Brains were dissected from postnatal mice euthanized by intraperitoneal injection of sodium pentobarbitone solution (200 mg/kg) (Euthatal; Merial Animal Health Ltd, UK). Dissected brains were rinsed in ice cold phosphate buffered saline (PBS), where necessary they were bisected in sagittal or coronal orientation before fixing in ice cold paraformaldehyde (PFA, 4% w/v in PBS) or methanol:acetic acid (3:1). Tissues were fixed overnight at 4 • C. PFA fixed brains were either embedded in wax or frozen in OCT. For wax embedding, PFA fixed brains were washed in PBS (1 h), dehydrated through an ethanol series (1 h each in 30%, 50%, 70%, 80%, 90%, 95%, 100% v/v), followed by isopropanol (1 h) and cleared in toluene (2 × 30 min). Methanol:acetic acid fixed tissues were washed in 70% ethanol and dehydrated and cleared as described above. Cleared brains were infiltrated with paraffin-wax at 58 • C (Fibrowax™, VWR International, Leuven) (2 × 12-24 h). Brains were orientated in paraffinwax filled moulds, allowed to set and blocks stored at 4 • C until sectioned.
PFA fixed brains for cryosectioning were washed in PBS (1 h), transferred to sucrose solution (30% w/v in PBS with azide 0.05% w/v) and allowed to sink at 4 • C (approximately 1-4 days). Brains were transferred to a 1:1 solution of sucrose:OCT™ (Optimal Cutting Temperature Compound, VWR, UK) at 4 • C (30 min). Tissues were placed in moulds containing OCT™ and frozen on a metal plate cooled on dry ice. Once frozen, tissue blocks were stored at −80 • C until sectioned.

Immunohistochemistry
Sections of paraffin embedded brains were dewaxed in Histoclear™ or Xylene (2 × 5 min). Slides were rehydrated through decreasing ethanol series (2 × 5 min: 100%, 3 min: 95%, 75%, 50%, 30%), washed in water (5 min) and PBS (5 min). Frozen sections were warmed to room temperature and washed in PBS (5 min) to remove OCT. If required, antigen retrieval was carried out by placing sections in antigen retrieval solution (Vector Laboratories) and microwaved until boiling for 20 min, after which they were allowed to cool (20 min). After antigen retrieval, slides were briefly dried, sections circumscribed with ImmEdge PAP pen (Vector laboratories Ltd, UK) and incubated in blocking buffer at room temperature (1 h) followed by incubation with primary antibodies (see Supplementary Material, Table S2 for list of primary antibodies) overnight at 4 • C. Slides were washed in PBS with Tween-20 0.1% (v/v) (PBST, 4 × 10 min). Appropriate secondary antibodies (Supplementary Material, Table S2) were used at a dilution of 1:1000 in blocking buffer and incubated for 1 h at RT. In all cases 4 ,6-Diamidino-2-Phenylindole (DAPI) was also added to secondary antibody mixture (1 μg/ml). Slides were washed in PBST (2 × 10 min) followed by PBS (2 × 10 min) and coverslips mounted with Mowiol (Polysciences Inc., Germany).

BrdU administration and detection
Bromodeoxyuridine (BrdU, 20 mg/ml in PBS) was administered to mice by intraperitoneal injection (100 mg/kg bodyweight) 1 h prior to euthanasia. Brains were dissected, fixed in methanol: acetic acid and processed for wax embedding. Paraffin sections were cut and dewaxed overnight in xylene and rehydrated. Sections were incubated in HCl (0.1 M) containing Pepsin (0.01% w/v) at room temperature (20 min). Slides were washed in PBS (2 × 10 min), slides briefly dried and sections circumscribed with PAP pen. BrdU incorporation was detected by immunohistochemistry using the monoclonal antibody G3G4 to BrdU (Supplementary Material, Table S2).

Image acquisition and analysis
Brightfield images were acquired with DMRB microscope using Leica DFC490 camera and Leica Application Suite (LAS) software. Low power brightfield images were obtained with Leica WILD MZ8 stereomicroscope using Leica DFC490 camera. Fluorescence images were acquired with DM5500B microscope equipped with motorized stage and Leica DFC360FX camera. Depending on the tissue thickness or specific stain either single images or Z-stacks were acquired using LAS software. LAS 3D-deconvolution algorithm was applied to Zstacks. Following deconvolution, either a single plane of focus was selected or Z-stacks were merged to create a maximum intensity projection. Comparisons were always made between images acquired and processed identically.

Image processing
Fiji imaging software was used to estimate length, area and cell/cilia numbers (68). Data were subsequently exported to Microsoft Excel or Minitab17 for further analysis. Fiji software was also used to 'stitch' large composite pictures from overlapping f luorescence images. Sequential images of equal exposure were taken in a grid across the region of interest with adjacent images having a small overlap. Software aligned identical overlapping regions to stitch images together. Brightfield and f luorescence images were processed using Adobe Photoshop software to adjust brightness. Adjustments were always applied to whole images and equally between images to be compared.

Statistical analysis
Statistical analysis was completed using Minitab17 software. Results were tested for normal distribution using Anderson Darling test and scores with P > 0.05 were deemed normal. The Bonett's test, a modified version of Lavard's test, and was used to determine variance of the mean and scores with P > 0.05 were deemed to have equal variance. Normal data with equal variance were analyzed using parametric tests; typically, a Student's t-test was used for comparison of means. Data that did not have either a normal distribution or equal variances were transformed to improve the distribution. If transformation was successful, parametric analyses were completed on transformed data, however if data still violated these assumptions non-parametric analyses were completed; typically, a Mann-Whitney U test was used for comparison of median values.

Quantification of primary cilia
The number of cells with primary cilia were identified by immunostaining for adenylyl cyclase III (ACIII). Nuclear DAPI labelling was used to quantify the total number of cells and the number of cells with a primary cilium expressed as a percentage. A region of interest (ROI) of 50 μm width was selected spanning a cross-section of both GCL and SGZ in the hippocampus. ROIs utilized the full thickness of the section (a Z-stack of 20 μm depth). Primary cilia were counted in ROIs from three sections of the brain per mouse and an average value was calculated. Average values from three mice were then used to make comparisons (n = 3). Data were normal with equal variance and a Student's t-test was used to show significance between means.

Analysis of Hippocampal thickness
The thickness of the GCL and SGZ was quantified by taking five equidistant measurements for each section and an average thickness obtained. Data from three brain sections from each mouse were used to calculate an average and three mice were used for comparisons (n = 3). Data were normal with an equal variance, so a Student's t-test was used to compare mean values.

Hippocampal cell type and analysis of proliferation
Hippocampal cell type and proliferation was quantified from sections co-immunostained for Paired box protein (Pax-6) and Neuronal nuclei (NeuN) or mini-chromosome maintenance protein 2 (MCM2) and marker of proliferation MKI67 (Ki67). The total number of labelled cells per 50 000 μm 2 was counted in the GCL and SGZ. Data were acquired from three brain sections per mouse and used to calculate an average. Average values from three mice were used for comparisons (n = 3). All data were normal with equal variance and was compared using a Student's t-test.
The proportion of Ki67 and MCM2 cells was compared by expressing the number of single-and doublelabelled cells as a percentage of total labelled cells for each section. Data were acquired from three brain sections per mouse and used to calculate an average. Average values from three mice were used for comparisons (n = 3). Percentages were normal with equal distribution and Student's t-test was used to compare mean values. Ki67-positive cells with GFAP-positive fibres were identified using high magnification images of individual cells through a 20 μm z-stack. The number of single-and double-positive cells were counted. The total number of labelled cells pooled to calculate the final percentage. Due to the low numbers of cells, statistical tests were not performed.

Quantification of misplaced Hippocampal neurons
NeuN-positive neurons positioned outside of the dentate gyrus were identified in a ROI encompassing the entire length of the GCL and extending 50 μm into the ML. The number of NeuN-positive cells were quantified and expressed as number of cells per 1000 μm. Data were acquired from three brain sections per mouse and used to calculate an average. Average values from three mice were used for comparisons (n = 3). Data were normally distributed with equal variance and a Student's t-test was used to compare means.

Isolation of hippocampal cells for neurosphere culture
Five day old pups were generated by crossing Talpid3fl/ fl;UbcCreERT2 stud males to Talpid3 fl/fl females. Brains were dissected from P5 mice and placed in DMEM/F-12 media (Life Technologies, UK). For generating hippocampal neurospheres, each brain was cut into coronal slices, transferred to DMEM/F12 media and dorsal hippocampi were isolated and incubated in 2 ml of digestion solution (1 mg/ml papain, 0.25 mg/ml L-cysteine, 1.1 mm EDTA,0.6% Glucose in PBS) at 37 • C for 15 min.
The genotype of each neurosphere culture was determined by PCR of DNA isolated from the spare tissue of dissected individual brains from which the line was derived. Experimental data were acquired from three Talpid3f l/f l;UbcCreER T2 hippocampal neurosphere lines isolated from three different mice (referred to as # 1, 2, 3).

Passaging of cultured neurospheres
Neurospheres were passaged by pH-disassociation using alkaline DMEM/F12 (pH 11.5) every 7 days and single cells resuspended in fresh culture media. Cell suspension was neutralized with acidic DMEM/F-12 (pH 2.0) and passed through a sieve (40 μm) to get rid of cell clumps. Cell viability was determined by Trypan blue dye exclusion. Cells were centrifuged and cell pellets were re-suspended in neurosphere culture media and plated in bacteriological petri dishes or 96 well plates (5000 cells per cm 2 , typically a 1:10 split).

Tamoxifen administration to cell cultures and genotyping to assess deletion of exons 11 and 12
All experiments for inducible deletion of Talpid3 were performed on Talpid3 fl/fl cells and Talpid3f l/f l;UbcCreERT2 at passage 3. Disassociated neurospheres were plated in bacteriological dishes or 96-well tissue cultures plates in culture media containing 1 μm 4-hydroxytamoxifen (4-OHT) and this was followed by an additional dose of 1 μm after 3 days of growth.
DNA was isolated from neurospheres of each genotype either with or without tamoxifen administration using the HotSHOT cell lysis method (69). The neurosphere lines were genotyped for the Talpid3 fl and Talpid3 exon 11-12 deleted alleles and for Cre using primers listed in Supplementary Material, Table S1.

Colony forming efficiency and colony size comparison
Twelve wells of a 96 well plate were plated with 1600 cells/well of each of the three independent Talpid3fl/fl; UbcCreER T2 cell lines (#1, 2, 3) to compare the effect of deletion of Talpid3 upon administration of Tamoxifen. This was followed by addition of media alone or media supplemented with tamoxifen. Media changes were made every third day. Control cells of genotype Talpid3 fl/fl were also compared with and without tamoxifen administration.
For estimation of colony size, 7-day neurospheres cultured in petri dishes either with or without administration of tamoxifen were disassociated into single cells. After disassociation they were plated in 96-well tissue culture plate as described above without any further administration of tamoxifen. These are referred to as 'passage +1'.
To measure colony size, a single image was acquired of a colony at the centre of each of the twelve wells using Leica DMIL microscope equipped with Leica EC3 camera. The area of every visible colony was measured using Fiji imaging software and all measurements taken from the 12 wells were pooled. Colony areas did not have a normal distribution and transformation was unable to normalize the data. Median values were compared using a Mann-Whitney U test.

Neurosphere migration assay
Neurospheres of similar size were selected from 7-day cultures with and without 4-OHT treatment. Single neurospheres were plated in each well of a 24-well tissue culture dish coated with matrigel containing differentiation media (1 ml). Neurospheres were imaged after 2, 24, 48 and 72 h using Leica DMIL microscope equipped with Leica EC3 camera. The total neurosphere area was measured from images using Fiji imaging software. Twelve neurospheres from three independent lines of each genotype were used to make comparisons and results pooled between cell lines of the same genotype (n = 36). Data did not have an equal variance and could not be equalized by transformation, so a Mann-Whitney test was used to show significance between median values.

Immunohistochemistry of cultured cells
Fixed cells on coverslips were transferred into a fresh 24well culture plate containing PBS (10 min). PBS was aspirated and PBT-block was added (500 μl, appendix 1.28). Cells were allowed to block at room temperature (1 h), block was aspirated and primary antibodies (see Table  S2) diluted in PBT-block were added (100 μl). Primary antibodies were allowed to incubate overnight at 4 • C. Primary antibodies were removed and PBST added (500 μl) to wash coverslips (10 min). PBST was aspirated and the wash repeated a further three times. PBST was aspirated and appropriate secondary antibodies (see Table S3) and DAPI diluted in PBT-block were added (100 μl). Coverslips were incubated at room temperature (1 h) after which secondary antibodies were removed. PBST was added (500 μl) to wash coverslips (10 min), PBST was aspirated and wash repeated once more. PBS was then added (500 μl, 10 min), PBS was aspirated and wash repeated once more. A small drop of Mowiol (∼20 μl) was placed on a microscope slide and coverslips were mounted with cells facing down. Once dry, coverslips were sealed with nail varnish and imaged.

Staining for F-actin with Phalloidin
F-actin was identified by following the initial steps of After the first PBT-block Phalloidin conjugated to fluorescence Alexa Fluor ® 488 Phalloidin (Life Technologies, UK) was diluted 5 μl per 200 μl of PBS with BSA (1% w,v) according to manufacturer's instructions. DAPI was also added to the Phalloidin-488 solution, added to coverslips and incubated at room temperature (30 min). Coverslips were washed with PBS (4 × 10 min) and mounted with Mowiol.

Quantification of ciliated cells
Neurosphere cultures plated on coverslips were immunestained for adenylyl cyclase III and acetylated α-tubulin to identify primary cilia. Two coverslips from each cell line were stained and four random ROIs were imaged from each coverslip. The number of ciliated cells in each ROI was quantified and expressed as a percentage of total cell number. Values were pooled between cell lines and used for comparison between conditions (n = 24). Data were normally distributed with equal variance and a Student's t-test was used to compare mean values.

Supplementary Material
Supplementary Material is available at HMGJ online.